We are continuing investigation of the control mechanisms regulating DNA replication in Escherichia coli and mouse leukemia L5178Y cells. We have examined the ability of temperature sensitive initiation mutants to initiate premature rounds of synthesis, in the presence and absence of protein synthesis, after being held at non-permissive temperature for 1.5 generations. These studies suggest that it may be possible to initiate up to three rounds in the absence of protein synthesis, and that the primary effect of protein synthesis inhibition may be on chain elongation. The role of the nuclear membrane of mouse leukemia L5178Y cells in DNA replication was also examined by comparing the association of newly replicated DNA and total DNA with the nuclear membrane at various times during the S period. The results obtained indicate that there is no preferential association of newly replicated DNA to the nuclear membrane during initiation or chain elongation. Some evidence suggests that the DNA which is associated with the membrane may be rich in AT content and is possibly satellite DNA.